The ability to identify small molecule ligands for any protein of interest has far-reaching implications, both for the elucidation of protein function and for the development of novel pharmaceuticals. With the introduction of split-pool strategies for synthesis (Furka et al., Int. J. Pept. Protein Res. 1991, 37, 487; Lam et al., Nature 1991, 354, 82; each of which is incorporated herein by reference) and the development of appropriate tagging technologies (Nestler et al., J. Org. Chem. 1994, 59, 4723; incorporated herein by reference), chemists are now able to prepare large collections of natural product-like compounds immobilized on polymeric synthesis beads (Tan et al., J. Am. Chem. Soc. 1998, 120, 8565; incorporated herein by reference). These libraries provide a rich source of molecules for the discovery of new protein ligands.
With such libraries in hand, the availability of efficient methods for screening these compounds becomes imperative. One method that has been used extensively is the on-bead binding assay (Lam et al., Chem. Rev. 1997, 97, 411; incorporated herein by reference). An appropriately tagged protein of interest is mixed with the library and beads displaying cognate ligands are subsequently identified by a chromagenic or fluorescence-linked assay (Kapoor et al., J. Am. Chem. Soc. 1998, 120, 23; Morken et al., J. Am. Chem. Soc. 1998, 120, 30; St. Hilare et al., J. Am. Chem. Soc. 1998, 120, 13312; incorporated herein by reference). Despite the proven utility of this approach, it is limited by the small number of proteins that can be screened efficiently. In principle, the beads can be stripped of one protein and reprobed with another; however, this serial process is slow and limited to only a few iterations. In order to identify a specific small molecule ligand for every protein in a cell, tissue, or organism, high-throughput assays that enable each compound to be screened against many different proteins in a parallel fashion are required. Although Brown et al. (U.S. Pat. No. 5,807,522; incorporated herein by reference) have developed an apparatus and a method for forming high density arrays of biological macromolecules for large scale hybridization assays in numerous genetic applications, including genetic and physical mapping of genomes, monitoring of gene expression, DNA sequencing, genetic diagnosis, genotyping and distribution of reagents to researchers, the development of a high density array of natural product-like compounds for high-throughput screening has not been achieved.
Clearly, it would be desirable to develop methods for generating high density arrays that would enable the screening of compounds present in increasingly complex natural product-like combinatorial libraries in a high-throughput fashion to identify small molecule partners for biological macromolecules of interest.
The present invention provides compositions and methods to facilitate the high-throughput screening of compounds for the identification of desirable properties or interactions. In a preferred embodiment, the present invention provides compositions and methods to facilitate the identification of compounds that are capable of interacting with a biological macromolecule of interest. In one aspect, a composition is provided that comprises an array of more than one type of chemical compounds attached to a solid support, wherein the density of the array of compounds comprises at least 1000 spots per cm2, more preferably at least 5000 spots per cm2, and most preferably at least 10,000 spots per cm2. In another aspect, a composition is provided that comprises a plurality of one or more types of non-oligomeric chemical compounds attached to a glass or polymer support, wherein the density of the array of compounds comprises at least 1000 spots per cm2. In a particularly preferred embodiment, the chemical compounds are non-peptidic and non-oligomeric. In particularly preferred embodiments, these compounds are attached to the solid support through a covalent interaction. In another particularly preferred embodiment, small molecules are attached to the solid support through a covalent interaction. In a particularly preferred embodiment, the compounds are attached to the solid support using a Michael addition reaction. In another preferred embodiment, the compounds are attached to the solid support using a silylation reaction. In general, these inventive arrays are generated by: (1) providing a solid support, wherein said solid support is functionalized with a selected chemical moiety capable of interacting with a desired chemical compound to form an attachment; (2) providing one or more solutions of one or more types of compounds to be attached to the solid support; and (3) delivering said one or more types of compounds to the solid support whereby an array of compounds is generated and the array comprises a density of at least 1000 spots per cm2 (FIG. 1). In other embodiments, the array comprises a density of at least 5000 stops per cm2, and more preferably at least 10,000 spots per cm2.
In another aspect, the present invention provides methods for utilizing these arrays to identify small molecule partners for biological macromolecules (e.g., proteins, peptides, polynucleotides) of interest comprising: (1) providing an array of one or more types of compounds (e.g., more preferably, small molecules), wherein the array has a density comprising at least 1000 spots per cm2; (2) contacting the array with one or more types of biological macromolecules of interest; and (3) determining the interaction of specific small molecule-biological macromolecule partners (FIG. 1). In particularly preferred embodiments, the biological macromolecules of interest comprise a collection of one or more recombinant proteins. In another preferred embodiment, the biological macromolecules of interest comprise a collection of macromolecules from a cell lysate. In another preferred embodiment, the biological macromolecules of interest comprise a polynucleotide.
Unless indicated otherwise, the terms defined below have the following meanings:
xe2x80x9cAntiligandxe2x80x9d: As used herein, the term xe2x80x9cantiligandxe2x80x9d refers to the opposite member of a ligand/anti-ligand binding pair. The anti-ligand may be, for example, a protein or other macromolecule receptor in an effector/receptor binding pair.
xe2x80x9cCompoundxe2x80x9d: The term xe2x80x9ccompoundxe2x80x9d or xe2x80x9cchemical compoundxe2x80x9d as used herein can include organometallic compounds, organic compounds, metals, transitional metal complexes, and small molecules. In certain preferred embodiments, polynucleotides are excluded from the definition of compounds. In other preferred embodiments, polynucleotides and peptides are excluded from the definition of compounds. In a particularly preferred embodiment, the term compounds refers to small molecules (e.g., preferably, non-peptidic and non-oligomeric) and excludes peptides, polynucleotides, transition metal complexes, metals, and organometallic compounds.
xe2x80x9cLigandxe2x80x9d: As used herein, the term xe2x80x9cligandxe2x80x9d refers to one member of a ligand/anti-ligand binding pair, and is referred to herein also as xe2x80x9csmall moleculexe2x80x9d. The ligand or small molecule may be, for example, an effector molecule in an effector/receptor binding pair.
xe2x80x9cMichael Additionxe2x80x9d: The term xe2x80x9cMichael additionxe2x80x9d refers to the reaction in which compounds containing electron-rich groups (e.g, groups containing sulfur, nitrogen, oxygen, or a carbanion) add, in the presence of base, to olefins of the from C=Cxe2x80x94Z (including quinones), where Z is an electron-withdrawing group, such as aldehydes, ketones, esters, amides, nitriles, NO2, SOR, SO2R, etc.
xe2x80x9cMicroarrayxe2x80x9d: As used herein, the term xe2x80x9cmicroarrayxe2x80x9d is a regular array of regions, preferably spots of small molecule compounds, having a density of discrete regions of at least about 1000/cm2.
xe2x80x9cNatural Product-Like Compoundxe2x80x9d: As used herein, the term xe2x80x9cnatural product-like compoundxe2x80x9d refers to compounds that are similar to complex natural products which nature has selected through evolution. Typically, these compounds contain one or more stereocenters, a high density and diversity of functionality, and a diverse selection of atoms within one structure. In this context, diversity of functionality can be defined as varying the topology, charge, size, hydrophilicity, hydrophobicity, and reactivity to name a few, of the functional groups present in the compounds. The term, xe2x80x9chigh density of functionalityxe2x80x9d, as used herein, can preferably be used to define any molecule that contains preferably three or more latent or active diversifiable functional moieties. These structural characteristics may additionally render the inventive compounds functionally reminiscent of complex natural products, in that they may interact specifically with a particular biological receptor, and thus may also be functionally natural product-like.
xe2x80x9cPeptidexe2x80x9d: According to the present invention, a xe2x80x9cpeptidexe2x80x9d comprises a string of at least three amino acids linked together by peptide bonds. Peptide may refer to an individual peptide or a collection of peptides. Inventive peptides preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide maybe modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
xe2x80x9cPolynucleotidexe2x80x9d or xe2x80x9coligonucleotidexe2x80x9d: Polynucleotide or oligonucleotide refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2xe2x80x2-fluororibose, ribose, 2xe2x80x2-deoxyribose, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5xe2x80x2-N-phosphoramidite linkages).
xe2x80x9cSmall Moleculexe2x80x9d: As used herein, the term xe2x80x9csmall moleculexe2x80x9d refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are xe2x80x9cnatural product-likexe2x80x9d, however, the term xe2x80x9csmall moleculexe2x80x9d is not limited to xe2x80x9cnatural product-likexe2x80x9d compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight of less than 1500, although this characterization is not intended to be limiting for the purposes of the present invention. Examples of xe2x80x9csmall moleculesxe2x80x9d that occur in nature include, but are not limited to, taxol, dynemicin, and rapamycin. Examples of xe2x80x9csmall moleculesxe2x80x9d that are synthesized in the laboratory include, but are not limited to, compounds described in Tan et al., (xe2x80x9cStereoselective Synthesis of over Two Million Compounds Having Structural Features Both Reminiscent of Natural Products and Compatible with Miniaturized Cell-Based Assaysxe2x80x9d J. Am. Chem. Soc. 1998, 120, 8565) and pending application Ser. No. 08/951,930 xe2x80x9cSynthesis of Combinatorial Libraries of Compounds Reminiscent of Natural Productsxe2x80x9d, the entire contents of which are incorporated herein by reference. In certain other preferred embodiments, natural-product-like small molecules are utilized.